Andres Buonanno, PhD, Head, Section on Molecular Neurobiology
Detlef Vullhorst, PhD, Staff Scientist
Irina Karavanov, PhD, Biologist
Carmen Gonzalez, PhD, Visiting Fellow
Oh-Bin Kwon, PhD, Visiting Fellow
Joerg Neddens, PhD, Visiting Fellow
Leqin Yan, PhD, Visiting Fellow
Zaheer Rana, MS, Predoctoral Fellow ¹

How experience and genes interact to remodel the nervous system during development is a central question in biology with major implications for disease. The goal of our laboratory is to investigate the molecular mechanisms that regulate neuronal and muscle plasticity during development in response to experience. We are studying how neuregulin signaling, via its tyrosine kinase receptors (ErbB 2-4), regulates synaptic plasticity in the brain and modulates behavior. Understanding this signaling is important for two reasons: (1) numerous clinical studies have identified associations between polymorphisms in both neuregulin-1 and ErbB-4 genes and a susceptibility for schizophrenia; and (2) NRG/ErbB-4 signaling is altered in postmortem brains of persons diagnosed with schizophrenia. Our laboratory also focuses on the identification and characterization of transcription factors that modify contractile properties of skeletal muscles both during development and in response to different types of activity in the adult, such as exercise.

Neuregulin-1 regulates synaptic plasticity: possible relevance to schizophrenia

Kwon, Longart, ² Gonzalez, Vullhorst, Buonanno; in collaboration with Hoffman

Neuregulin-1 (NRG-1), a trophic and differentiation factor harboring an EGF-like domain, signals via the ErbB family of receptor tyrosine kinases. Although NRG-1 function has been extensively studied in the developing peripheral nervous system, little was known until recently about its role in the developing and adult brain. Previously, we demonstrated that ErbB and NMDA receptors (NMDARs) co-localize at glutamatergic post-synaptic densities and that ErbB-4 directly interacts with scaffolding proteins, such as PSD-95, that are important regulators of synaptic plasticity. Based on these findings, we proposed that NRG/ErbB signaling modulates activity-dependent synaptic plasticity. Our recent work supports our hypothesis.

We found that NRG-1 reverses ("depotentiates") long-term potentiation (LTP) at hippocampal CA1 glutamatergic synapses in an activity-dependent fashion. Inhibitors that selectively target ErbB tyrosine kinases block NRG-1-dependent depotentiation and raise LTP levels at already potentiated synapses. Using patch clamp and cell-biological techniques, we demonstrated that NRG-1 depotentiates LTP by selectively reducing AMPA, but not NMDA, receptor currents. Live imaging of hippocampal neurons transfected with AMPA receptors fused to superecliptic green fluorescent protein (seGFP), a form of GFP that fluoresces strongly only when expressed on the cell surface, indicates that NRG-1 stimulates the internalization of surface seGluR1-containing AMPA receptors. Consistent with these and our earlier findings, others have shown that NRG-1 and ErbB receptor hypomorphic mice develop normally but exhibit a reduction in glutamate receptor levels and manifest behavioral deficits. This novel regulation of LTP by NRG-1 has important implications for the modulation of synaptic homeostasis at glutamatergic synapses, which can affect cognition, learning, and memory, and for understanding molecular mechanisms that underlie complex disorders such as schizophrenia.

Regulation of ErbB-4 surface clustering by PSD-95 at inhibitory hippocampal neurons

Chatani-Hintze, ³ Longart, ² Gonzalez, Vullhorst, Yan, Buonanno

To extend our earlier work, showing that ErbB-4 directly interacts with the post-synaptic density protein PSD-95 at glutamatergic synapses, we investigated the developmental expression and trafficking of the receptor. These interactions may be of special interest because they appear to be altered in postmortem brain tissue isolated from persons diagnosed with schizophrenia. Using immunofluorescence analysis in hippocampal slices and dissociated neurons in culture, we found that ErbB-4 receptors are expressed predominantly at glutamatergic synapses in GABAergic interneurons. We investigated the trafficking of ErbB-4 in cultured hippocampal neurons by surface protein biotinylation and antibody labeling of receptors in live cells. We found that ErbB-4 immunoreactivity in developing neurons precedes PSD-95 expression, with ErbB-4 clusters initially forming in the absence of, but later associating with, PSD-95-positive puncta. The surface fraction of dendritic ErbB-4 increases from 30 percent at six days in culture to 65 percent by day 16. Interestingly, receptor activation by NRG-1 triggers significant internalization in young and mature neurons despite increased association of ErbB-4 with PSD-95. PSD-95 primarily seems to control receptor clustering. These findings enhance our understanding of the role of ErbB-4/PSD-95 protein interaction for NRG-mediated signaling at glutamatergic synapses.

Expression and function of the NMDA NR2C receptor in beta-galactosidase knockin mice

Buonanno, Karavanov; in collaboration with Lu, Vicini

We previously reported that expression of the NR2C subunit of the NMDAR is regulated by NRG-1 in cultured organotypic slices of cerebellum. NR2C has generally been considered a "cerebellar subunit" because its expression is strikingly higher in the cerebellum than in other brain areas. To study the precise expression and function of the NR2C subunit in the developing brain, we developed knockin mice. Using homologous recombination, we replaced DNA sequences encoding most of the NR2C protein with the E. coli nlacZ gene that encodes the beta-galactosidase (B-gal) reporter. By staining whole brains and sections with X-gal, we identified cells histologically that express the B-gal reporter under control of NR2C transcriptional regulatory elements. Using the knockin mice, we found that NR2C is more dynamically and broadly expressed in brain than previously reported. In the cerebellum, NR2C is expressed in a caudalrostral gradient and in a series of parasagittal bands in subsets of cerebellar granule cells. We also found NR2C expression in previously unappreciated areas, such as the retrosplenial and cerebral cortex, hippocampus, and basal ganglia. Using cell type-specific antibodies, we unexpectedly found that NR2C is expressed by glial cells, not neurons, dispersed in the hippocampus, striatum, olfactory bulb, and cerebral cortex. All novel sites of expression, identified in NR2C tg-nlacZ knockin mice, were confirmed by in situ hybridization using 33P-labeled NR2C cRNA probes. The expression of NR2C in discrete brain areas outside the cerebellum and in glia suggests that NMDARs with different subunit compositions may serve distinct functions.

In collaboration with members of the laboratories of Vicini and Wolfe, we studied the NMDAR excitatory post-synaptic currents (EPSCs) in solitary cerebellar neurons cultured in micro-islands from wild type (WT) and NR2C tg-nlacZ knockin mice and NR2A subunit knockout mice. NR2C tg-nlacZ granule neurons have larger NMDA-EPSCs than those of WT cells. The decay time of the currents was without exception unexpectedly fast, and the quantal content was higher in the mutant mice. The most striking result was a significant increase in the NMDA-EPSC peak amplitude and charge transfer in NR2C tg-nlacZ knockin mice, which is largely attributable to an increase in quantal size, as estimated from miniature NMDA-EPSCs.